Do matrix metalloproteinase and cathepsin K inhibitors work synergistically to reduce dentin erosion?

Abstract Objectives To evaluate the effects of matrix metalloproteinase (MMP) and cathepsin K (catK) inhibitors on resistance to dentin erosion. Methodology A total of 96 dentin specimens (3×3×2 mm) were prepared and randomly assigned into four groups (n=24): deionized water (DW); 1 µM odanacatib (ODN, catK inhibitor); 1 mM 1,10-phenanthroline (PHEN, MMP inhibitor); and 1 µM odanacatib + 1 mM 1,10-phenanthroline (COM). Each group was further divided into two subgroups for the application of treatment solutions before (PRE) and after erosive challenges (POST). All specimens were subjected to four daily erosive challenges for 5 d. For each erosive challenge, the specimens in subgroup PRE were immersed in the respective solutions before cola drinks, while the specimens in subgroup POST were immersed in the respective solutions after cola drinks (the immersion duration was 5 min in both cases). All specimens were stored in artificial saliva at 37°C between erosive challenges. The erosive dentin loss (EDL) was measured by profilometry. The residual demineralized organic matrix (DOM) of specimens was removed using type VII collagenase and evaluated by profilometry. Both the EDL and thickness of the residual DOM were statistically analyzed by two-way analysis of variance (ANOVA) and Bonferroni’s test (α=0.05). The surface topography and transverse sections of the specimens were observed using SEM. MMPs and catK were immunolabeled in the eroded dentin and in situ zymography was performed to evaluate the enzyme activity. Results Significantly lower EDL was found in the groups ODN, PHEN, and COM than in the control group (all p<0.05), while no significant difference in EDL was found among the groups ODN, PHEN, and COM (all p>0.05). The application sequence showed no significant effect on the EDL of the tested groups (p=0.310). A significantly thicker DOM was observed in the group ODN than in the control group regardless of the application sequence (both p<0.05). The treatment with ODN, PHEN, and COM inhibited the gelatinolytic activity by approximately 46.32%, 58.6%, and 74.56%, respectively. Conclusions The inhibition of endogenous dentinal MMPs and catK increases the acid resistance of human dentin but without an apparent synergistic effect. The inhibition of MMPs and catK is equally effective either before or after the acid challenge.


Introduction
Tooth erosion occurs by the dissolution of dental apatite when exposed to acids, without bacterial involvement, 1 with a global prevalence in adolescence estimated at 30%. 2 For tooth erosion in the initial stage, the effects of acids are limited to the enamel. 3 Exposure to acids results in the dissolution of the inorganic dental hard tissue, 3 which progressively leads to extensive loss of substance. 3 When enamel loss occurs, the effects of acids further extend into the dentin, and dentin erosion can occur. 3,4 During the progression of dentin erosion, minerals are released from the peritubular and intertubular dentin. 5 Thereafter, a layer of a fully demineralized organic matrix (DOM) is formed in the superficial dentin, followed by a partially demineralized zone and by the intact inner dentin. 4,5 DOM is regarded as a diffusion barrier against erosive acids and active ingredients released by demineralization, as well as a form of resistance to abrasive forces. 6,7 Furthermore, maintaining DOM is important for dentin remineralization, especially in the presence of calcium and phosphate together with fluoride. 8 However, DOM can be degraded by collagenases (e.g., matrix metalloproteinases (MMPs) and cysteine cathepsins (CCs)), leading to increased mineral loss in demineralized dentin and further accelerating the progression of dentin erosion. [8][9][10] In previous studies, the application of both MMP 5,11 and CC inhibitors 5 significantly reduced erosive dentin loss (EDL) by approximately 21-42%.
MMPs are mostly secreted as latent proenzymes (stated as proMMPs), in which the functional activity of the catalytic domain is inhibited by the prodomain. 12 When the prodomain is removed by other proteases, such as CCs, proMMPs can be activated. 12 Christensen and Shastri 13 (2015) reported that proMMP-9 was activated because the prodomain was cleaved by cathepsin K (catK) in vitro. Moreover, using odanacatib (a catK inhibitor) significantly reduced the release of cross-linked carboxyterminal telopeptide of type I collagen (ICTP, the specific products of type I collagen degraded by MMPs). 14 On the other hand, Li, et al. 15 (2004) found that MMP-1 cleaved type I collagen released 3/4 and 1/4 fragments, which allowed further degradation by CCs (e.g., catK, catB, and catL). Therefore, MMPs and CCs may synergistically contribute to the progression of dentin erosion. 5,13,15 In our previous study, 1,10-phenanthroline (an MMP inhibitor), E-64 (a CC inhibitor), and a combination of the two inhibitors were applied before erosion, aiming to reduce EDL. 5 In that study, EDL was significantly reduced and a thicker DOM was preserved compared to that of the control group. 5 However, no significant differences were found either in EDL or DOM between individual and combined applications. 5 This phenomenon may be related to the application sequence of MMP and catK inhibitors. Importantly, MMPs become functional after the acidic pH is neutralized by the saliva buffer, while CCs exhibit optimal functional activity at a slightly acidic pH (e.g., catK exhibits optimal activity at pH=5.5) and may be irreversibly inactivated at a neutral pH. 11 Moreover, a recent study showed that chlorhexidine performed better than sodium fluoride in controlling dentin erosion and, interestingly, chlorhexidine was more effective in reducing EDL when applied after erosion than when applied before erosion. Applying chlorhexidine to patients at risk of dentin erosion (e.g., in the event of gingival recession) before an acid invasion can be considered a pre-erosion application, whereas applying chlorhexidine to patients suffering from dentin erosion can be considered a post-erosion application. 16 It was also reported that sodium fluoride could inhibit MMPs and that chlorhexidine could inhibit MMPs as well as CCs. 17,18 Accordingly, a synergistic effect of MMP and CC inhibitors applied after erosion may exist. Furthermore, erosive attacks would result in a hollowing and funneling of the dentinal tubule. 19 Compared to prior treatment with an inhibitor, prior treatment with acid may increase the possibility of protease inhibitors penetrating dentin; thus, the application sequence of MMP and catK inhibitors may play an important role in their potential synergistic effect on resisting dentin erosion. However, the information available in the literature is scarce. Therefore, this study aimed to evaluate the effects of MMP and catK inhibitors on resisting dentin erosion.
The following null hypotheses were tested: 1) there was no difference in EDL and the thickness of residual DOM between the protease inhibitors; 2) there was no difference in EDL and the thickness of residual DOM between different application sequences of the protease inhibitors. Preparation of dentin specimens A total of 56 healthy human third molars that were freshly extracted from 18 to 30 years subjects, male and female, were collected and stored in 0.05% thymol solution at 4°C. 20 The molars were divided longitudinally into equal halves. Subsequently, separation was performed at the cementum-enamel junction (CEJ) using a low-speed diamond saw (Isomet, Buehler, Lake Bluff, USA) under water irrigation. The coronal enamel was then removed from each half of the tooth at the dentinal-enamel junction (DEJ) to expose the dentin. In total, 96 dentin blocks (3×3×2 mm) were extracted from the central region, located between the pulp horns of the coronal dentin (prepared from 50 molars). A microhardness tester with a Vickers diamond indenter was used to measure the initial microhardness of the dentin surface, ensuring that the dentin blocks were prepared from similar regions. A total of three indentations at 0.3 intervals on the dentin surface were created with a 100 gf load for 10 s. 21 The average of the three microhardness values within 63.67 to 67.16 kg/mm 2 was included. 21 The dentin blocks were further embedded in acrylic resin (Paladur, Heraeus Kulzer, Germany) with a 6 mm internal diameter and a 5 mm height. A baseline surface was finished by polishing the dentin surface using 220-, 600-and 1200-grit silicon carbide abrasive papers and cleaned in an ultrasound bath for two sessions of 1 min. Both sides of the baseline surface were covered with nail varnish (Revlon Corp., NY, USA) to generate a reference surface, and the middle area of the surface (3×1 mm) remained unaltered to receive erosive attacks. 22 All the specimens were stored in deionized water to maintain humidity.
In addition, six dentin slices (200 μm), parallel to the long axis of the teeth, were cut from six teeth (1 slice/tooth) and used to confirm the presence of MMPs and catK in dentin, as well as to show the enzyme activity before and after treatment with MMP and catK inhibitors.

Erosive challenges
All 96 specimens were randomly divided into four groups (n=24) according to the following treatment Before the erosive challenges, specimens were first soaked in artificial saliva (containing 0.4 g/l NaCl; 0.795 g/l CaCl 2 ·H 2 O; 0.4 g/l KCl; 0.005 g/l Na 2 S·9H 2 O; 0.69 g/l NaH 2 PO 4 ·H 2 O; 0.3 g/l KSCN; and 1 g/l urea, pH=6.8) at 37°C for 1 h. 16 In the subgroup PRE, the specimens were immersed in 50 ml of the respective solutions for 5 min, followed by a 10 s rinse with deionized water. 23 Subsequently, the specimens were immersed in 150 ml of a cola drink Profile measurement Nail varnish was carefully removed by a scalpel blade to expose the intact reference surface. 9 The first profile measurement was performed using a contact profilometer (SEF 680, Kosaka Laboratory, Japan) after the 5 d cycles. The surface of the dentin specimens was scanned using a stylus, the differences in five heights between the reference surface of the specimens and the eroded surface were measured, and the mean value for each specimen was estimated as the EDL (in μm). 5 During the measurement, the dentin samples were kept moist in deionized water to prevent DOM from shrinking. 4,22 After that, specimens were incubated in artificial saliva containing 100 U/ ml type VII collagenase from Clostridium histolyticum  Simple effect analysis was adopted when a significant interaction was found. 16 All statistical analyses were performed at a 0.05 significance level. Table 1 shows the EDL and residual DOM of different groups. The different protease inhibitors significantly affected the EDL and thickness of the residual DOM (both p<0.05). The application sequence did not exert a significant effect on the EDL of the tested groups (p>0.05), while the application sequence significantly affected the thickness of residual DOM (p<0.05). Regarding EDL, no significant interaction was found between the different protease inhibitors and application sequences (p=0.613). Regarding DOM, a significant interaction was found between the different protease inhibitors and application sequences (p=0.013).

Results
Significantly less EDL was observed in the groups ODN, PHEN, and COM than in the group DW (all p<0.05). No significant differences in EDL were detected among the groups ODN, PHEN, and COM (all p>0.05). A significantly thicker DOM was observed in the groups ODN and COM than in the group DW (both p<0.05). No significant differences were found in the DOM between the groups PHEN and DW (p=0.615).
A simple effect analysis was adopted since a significant interaction was found between the different protease inhibitors and application sequences

Discussion
Compared to enamel erosion, dentin erosion progresses much faster due to differences in mineral content and impurity, as well as the enzymatic degradation of collagen by endogenous MMPs and Different lowercase letters in a column indicate significant differences in different groups and subgroups (P<0.05). DW: deionized water; ODN: 1 µM odanacatib; PHEN: 1 mM 1,10-phenanthroline; COM: 1 µM odanacatib + 1 mM 1,10-phenanthroline; PRE, before the erosive challenges; POST, after the erosive challenges. Do matrix metalloproteinase and cathepsin K inhibitors work synergistically to reduce dentin erosion?
A, B: specimen treated with deionized water; C, D: specimen treated with 1 µM odanacatib; E, F: specimen treated with 1 mM 1,10-phenanthroline; G, H: specimen treated with 1 µM odanacatib + 1 mM 1,10-phenanthroline. catK in dentin. 5,19,27 To the authors' knowledge, this study was the first to investigate the synergistic effects of MMP and catK inhibitors on resisting dentin erosion by considering the application sequence.
Based on our findings, the null hypotheses of no difference in EDL between the protease inhibitors and no difference in EDL between the application sequence of protease inhibitors were accepted. The null hypotheses of no difference in the thickness of residual DOM between the protease inhibitors and no difference in the thickness of residual DOM between application sequences of protease inhibitors were rejected. The research protocol, specifically the regimen of erosive challenges, the application times, and the concentrations of the tested inhibitors were determined according to previous studies. 5,14,16,23 In addition, among all known CCs, catK is the only one with triple helical collagenase activity 14 and it has been indicated that pro-MMP-9 can be activated by catK. 13 Therefore, we used ODN in this study because it is a specific inhibitor of catK. 25 The application of MMP and catK inhibitors alone significantly reduced EDL, agreeing with previous studies. 5,11 Moreover, the application sequence did not compromise the effectiveness of MMP and catK inhibitors in reducing EDL. MMPs are a family of Zn 2+ -and Ca 2+ -dependent enzymes, and PHEN may inactivate MMPs by chelating Zn 2+ with two N atoms. 12,28 Furthermore, PHEN is a small-molecule 29 (molecular weight: 180.205) metal chelator; 30,31 thus, PHEN may remain in dentin and function regardless of the condition in which it was applied. Pertinently, catK is a cysteine proteinase, containing a thiol group (-SH). 32 ODN may combine with the -SH of catK and thus prevent catK from binding to its substrates. 32 The optimal pH of catK has a short duration, which may explain why catK inhibitors exhibited a similar effect when applied before and after erosion. 11 Interestingly, no synergistic effect of MMP and catK inhibitors on dentin erosion was detected in this study. Activated Despite the similar EDL in the subgroups PRE and POST, treatment with MMP and catK inhibitors after erosive challenges showed more numerous and wider dentinal tubules than that before erosive challenges, which might indicate more aggressive acid etching.
Interestingly, a similar DOM thickness was observed in the group treated with the MMP inhibitor and in the control group, which is in line with the findings from Zarella, et al. 11 (2015). However, contradictory results were reported by Yang, et al. 5 (2022). The contrasting results may be related to the different study protocols (e.g., erosion duration, application time, and types of inhibitors). MMPs lead to excessive DOM degradation after exposure to an acidic pH for a sufficient duration and neutralization by saliva A, B: specimen treated with deionized water; C, D: specimen treated with 1 µM odanacatib; E, F: specimen treated with 1 mM 1,10-phenanthroline; G, H: specimen treated with 1 µM odanacatib + 1 mM 1,10-phenanthroline. The demineralized organic matrix (DOM) is indicated by the dashed line. buffer. 11 With our protocol, MMPs may be insufficiently inhibited since the concentration of inhibitor necessary to effectively inhibit enzymatic activity is related to the application method (e.g., stronger inhibition occurred when MMP inhibitor was mixed with MMPs than when added to zymography buffer). 8 Applying the catK inhibitor and the combination of the two inhibitors significantly preserved a thicker DOM than that of the control group. In addition, a significantly thicker DOM was observed when the combined application was performed after erosion rather than before erosion.
Compared to treatment with an MMP inhibitor before erosion, higher MMP activity (11-17% higher) was detected when dentin specimens were treated with an MMP inhibitor following acid erosion. 8 As mentioned earlier, it is possible that catK plays an essential role in the cascade reaction of MMP activation. 12,13,34  Based on our findings, applying MMP and catK inhibitors is an effective method for reducing dentin erosion. An MMP or catK inhibitor can be applied before or after erosion, but the combined application is not strictly necessary. CatK inhibitors are considered a potentially promising solution for managing dentin erosion. The products tested in this study were all A, D: bright field, showing the optical density of eroded dentin surface; B: acquired in green channel, indicating immunolabeled MMP-8 in eroded dentin; E: acquired in red channel, indicating immunolabeled catK in eroded dentin; C, F: merged images. analytical-grade reagents (high purity) with acceptable costs. For clinical usage, it will be necessary to further identify a reasonable carrier for MMP and catK inhibitors, such as a gel or paste.
A limitation of this in vitro study is that the real state of the oral environment cannot be represented.
Moreover, only one concentration and duration of protease inhibitors were tested. No positive control was adopted since the study was performed to provide a proof of concept for the synergistic effects of MMP and catK inhibitors on dentin erosion. Commonly used anti-erosive agents, such as stannous fluoride and sodium fluoride, should be included as positive controls in further studies. In addition, further clinical studies are needed to confirm our findings.

Conclusions
Within the limitations of this study, the following conclusions can be prudently drawn: A, D, G, J: bright field, showing the optical density of the dentin surface; B, E, H, K: acquired in green channel, showing fluorescence in dentin; C, F, I, L: merged images. A-C: dentin slices treated with deionized water (DW, without inhibitory treatment); D-F: dentin slices treated with 1 µM odanacatib (ODN); G-I: dentin slices treated with 1 mM 1,10-phenanthroline (PHEN); J-L: dentin slices treated with 1 µM odanacatib + 1 mM 1,10-phenanthroline (COM). M: quantified green fluorescence of the specimens with different treatments. The intense green fluorescence indicated strong gelatinolytic activity in dentin without inhibitory treatment. Treatment with catK and MMP inhibitors led to decreased green fluorescence, indicating reduced gelatinolytic activity after inhibitory treatment. 1. The inhibition of endogenous dentinal MMPs and catK increases the acid resistance of human dentin but shows no apparent synergistic effect.
2. The inhibition of MMPs and catK either before or after the acid challenge is equally effective.
3. The inhibition of catK seems to be the simplest approach and least dependent on the application mode when the inhibition of endogenous dentinal collagenolytic enzymes is considered in the prevention of dentin erosion progression.